Cloning and expression of 45 kDa Taenia asiatica estrogen-regulated protein and development of monoclonal antibodies

doi:10.12140/j.issn.1000-7423.2019.01.013

Abstract

Abstract:

Objective To clone and express 45 kDa Taenia asiatica estrogen-regulated protein (TaEP45) as a diagnostic antigen for detecting infection of T. asiatica. Methods The open reading frame of TaEP45 was amplified from the total cDNA of T. asiatica using specific primers designed from TaEP45 sequence deposited in GenBank, then subcloned into the prokaryotic expression vector pET30a(+). The recombinant plasmid DNA was transformed into Escherichia coli BL21（DE3） cells for expression. The recombinant TaEP45 with His-tag was expressed in E. coli under induction of 1 mmol/L isopropyl β-D-thiogalactoside(IPTG) and purified with nickel column. The purified recombinant TaEP45 was tested for its recognition by serum from pig infected with T. asiatica by Western blotting, and then used to immunize BALB/c mice to make monoclonal antibody (McAb). Results RT-PCR amplified a gene product of 1 362 bp which encodes 453 amino acids of TaEP45. The recombinant TaEP45 was highly expressed in E. coli BL21 cells with relative molecular mass of 60 000. The purified recombinant TaEP45 was strongly recognized by serum from pig infected with T. asiatica. After being immunized with recombinant TaEP45 formulated with Freud’s adjuvant, the splenocytes of mice were used to make hybridoma cell lines. Total 5 hybridoma cell lines were obtained that stably produced McAbs against TaEP45 with titers higher than 1 : 2 430. The subtype antibodies were IgG2b or IgG1. All McAbs were able to recognize T. asiatica worm lysates and recombinant TaEP45 as well. Conclusion The TaEP45 was successfully cloned from T. asiatica cDNA and the recombinant TaEP45 protein was expressed in E. coli. Five hybridoma cell lines that stably secreted McAbs against TaEP45 are obtained with high titer of antibodies, which are able to recognize T. asiatica worm lysates.

Key words: Taenia asiatica, 45 kDa estrogen-regulated protein, Hybridoma cell strains

CLC Number:

R383.32

Li-qun WANG, Pan-hong LIANG, Shao-hua ZHANG, Jun-ling HOU, Li-jie WANG, Li MAO, Xue-nong LUO. Cloning and expression of 45 kDa Taenia asiatica estrogen-regulated protein and development of monoclonal antibodies[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2019, 37(1): 70-74.

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杂交瘤细胞株 Cell lines	培养上清不同稀释度的A₄₅₀值 A₄₅₀ value of different dilutions of culture supernatants						阴性对照 Negative control	抗体效价 Antibody titer	抗体亚型 Isotype of McAbs	抗体浓度 /μg·ml^-1 Antibody concentration /μg·ml^-1
杂交瘤细胞株 Cell lines	1 : 10	1 : 30	1 : 90	1 : 270	1 : 810	1 : 2 430	阴性对照 Negative control	抗体效价 Antibody titer	抗体亚型 Isotype of McAbs	抗体浓度 /μg·ml^-1 Antibody concentration /μg·ml^-1
2C11A3	2.590	1.464	2.226	1.696	0.993	0.461	0.069	1 : 2 430	IgG2b,κ	8.405
5C11D3	2.468	2.359	2.142	1.715	1.052	0.511	0.069	1 : 2 430	IgG2b,κ	8.843
4B95H	2.214	2.029	1.628	1.210	0.732	0.367	0.069	1 : 2 430	IgG1,κ	6.058
8E7D5	2.599	2.527	2.204	1.533	0.801	0.352	0.069	1 : 2 430	IgG1,κ	6.948
10E6D4	2.571	2.560	2.422	2.014	1.370	0.705	0.069	1 : 2 430	IgG2b,κ	8.704